Insulin binding to cells or detergent extracts will be measured before and after proteolytic and phospholipase treatment of the cell surface. These measurements combined with cell fractionation techniques will determine whether cryptic membrane or internal organelle related pools of receptor may be detected and selectively isolated from the large pool of receptor on the surface of the plasma membrane. By labelling the receptor with photoaffinity analogs of insulin or with dense amino acids, the turnover rates of receptor in the pools will be measured and a sequence for new receptor passage through each pool assigned. These procedures will establish normal steady-state rates for those aspects of receptor processing which include receptor synthesis, insertion into the plasma membrane, internalization, recycling, and degradation. Charge or size modifications that might occur during receptor processing will be analyzed by SDS-gel electrophoresis and isoelectric focusing of surface receptor photolabelled with iodinated insulin analogs. Insulin dependent changes in receptor-protein interactions in the plasma membrane also will be monitored using photoactivated bifunctional crosslinking agents. These will provide rapid light induced stabilization of receptor aggregate distribution for analysis after detergent extraction. All of the above parameters will be characterized in 3T3-L1 adipocytes and 3T3-C2 fibroblasts. Changes in these parameters will be compared to alterations in the cells biological response to insulin when normal receptor processing has been perturbed by events such as receptor "down-regulation", or by treatment with drugs or other metabolic inhibitors used in studies of receptor metabolism. These studies should more directly define the kinetics and routes of receptor processing than investigations to date. As 3T3-C2 fibroblasts suppress their level of receptors and lose sensitivity to insulin after prolonged exposure to the hormone, and 3T3-L1 adipocytes to not, the results of these studies could directly relate to obtaining a better understanding of the underlying mechanism defects in some insulin resistant disease states.